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INT JVR6-1.Qxd Iranian Journal of Veterinary Medicine Identification of Gyrodactylus gurleyi in Carassius auratus using morphometric and molecular characterization Omidzahir, Sh.1, Ebrahimzadeh Mousavi, H.A.1*, Soltani, M.1, Shayan, P.2, Ebrahimzadeh, E.2, Hoseini, M.3 1Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 2Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 3Department of Pathology, Faculty of Veterinary Medicine, Islamic Azad University Babol Branch, Babol,Iran. Key words: Abstract: Carassius auratus, Gyrodactylus gurleyi, BACKGROUNDS: Gyrodactylus is a small monogenean PCR, morphometric characterization, ectoparasite that lives on the skin and fins of most of the world's fish molecular characterization. species. Gyrodactylus appears to be one of the most prevalent parasites found in ornamental fish, especially in Cyprinids. Correspondence Goldfish (Carassius auratus) are a popular ornamental fish that are Ebrahimzadeh Mousavi, H.A. highly contaminated by Gyrodcatylus. OBJECTIVES: The present Department of Aquatic Animal Health, study is aimed to identify morphological and molecular character- Faculty of Veterinary Medicine, University istics of the Gyrodactylus parasite on gold fish. METHODS: The of Tehran, Tehran, Iran. morphological identification of Gyrodactylus specimens was Tel: +98(21) 61117001 performed using the measurements and drawings of opisthaptoral Fax: +98(21) 66933222 hard parts of the parasites. The molecular species description was Email: [email protected] based on a polymerase chain reaction (PCR) of partial sequence of the 5.8S region of ribosomal RNA, and a partial sequence of the Received: 3 November 2011 internal transcribed spacer 2 (ITS2) of ribosomal RNA. The Accepted: 14 February 2012 nucleotide sequences of the PCR products were compared with corresponding sequencing registered in GenBank. RESULTS: Based on the morphometric analysis and sequencing, the Gyrodactylus specimens were described as Gyrodactylus gurleyi. CONCLUSIONS: A combination of molecular techniques with morphological analysis seems to be the best approach for the identification of Gyrodactylus speices. Introduction and necessary. The Gyrodactylus (Gyrodactylidae: Monogenea: Carassius auratus is a freshwater fish, in the Platyhelminthes) is a small monogenean ectoparasite family of Cyprinidae, known under the common (<1 mm) which lives mainly on the skin and fins of name of goldfish. It is one of the most commonly kept freshwater and marine fish (Collins et al., 2002). aquarium fish. There are many different varieties of Ectoparasites of the genus Gyrodactylus can infect the domesticated goldfish. A large number of different most of the world's fish species. Gyrodactylus is one fish species, particularly goldfish, are annually of the parasite organisms that can cause diseases with imported to Iran from the Far-East (China), Russia high mortality rate, (Bakke et al., 2002; Meinila et al., and Eastern Europe (Shamsi et al., 2009). Also, 2004). Gyrodactylus parasites damage the host's goldfish are cultured and reproduced in several fish epidermis during attachment to the fish. Lesions in farms in Iran. Considering the economic importance the epidermis are caused by the 16 marginal hooks and interest of people for this ornamental fish, the and two anchors of the attachment organ. Ulcers study dealing whit the identification of pathogenic generated by enzymatic digestion cause loss and parasites for aquarium fish seem to be both important inability to osmoregulate which seems to be the main IJVM (2012), 6(1):41-46 41 Identification of Gyrodactylus gurleyi in... Ebrahimzadeh Mousavi, H.A. reason of the host mortality. The epidermal damage SSC-DC80P NO.401182)under the light microscope. caused by bacteria or fungi give rise to the secondary Photographic images of parasites were used for infections that can play a considerable role in the morphological analysis. After taking photos, each pathogenicity of the Gyrodactylus (Collins et al., Gyodactylus sample was drawn and measured. 2002). It is estimated that there are more than 20,000 Morphological identification of Gyrodactylus species of Gyrodactylus (Bakke et al., 2007). Alittle species was performed using features of opisthaptoral more than four hundred valid Gyrodactylus species characters (anchors, marginal hooks, ventral and are described, from nearly 400 hosts in fish families. dorsal bars) according to exiting Gyrodactylus So, identification of 20-30 new Gyrodactylus species identification keys. The characters and measurement per decade is a very low increased number to be criteria described in this study have been taken from identified in every ten year period (Harris et al., 2004; Malmberg (1970) (Fig 1). Then, each Gyrodactylus Bakke et al., 2002). specimen was removed from the fish under the light Malmberg 1970 identified the morphological microscope, and was individually preserved in the species of the Gyrodactylus based upon its sterile tube containing 70% (v/v) ethanol. opisthaptoral hard parts. In particular, the shape of the DNA extraction and PCR amplification: DNA tiny marginal hook sickles is species specific, but, was extracted by digesting a single parasite specimen unfortunately, the morphology of the attachment organ using a DNAisolation kit (MBST, Iran) according to is variable. Factors like host, geographical location, the manufacturer's instructions. and temperature impress the haptors intraspecific Polymerase chain reaction (PCR) amplification phenotypic variation (Rokicka et al., 2007). was performed using sense primer (5`-CGA TCA To resolve the problems of the identification of the TCG GTC TCTCGAAC-3`) at position 687 to 706 of Gyrodactylus specimens, molecular methods were the 5.8S rRNA gene, and the antisense primer (5`- introduced by Cunningham, et al., 1995. Molecular TTAAGG AAG AAC CAC TAG AG-3`) at position methods result in giving high quality systematic 1042 to 1061 of the ITS2 rRNAgene. information, that is mostly based on nuclear ITS in The amplification reaction contained 15 μl DNA Gyrodactylus (Ziêtara et al., 2008; Matìjusová et al., sample, 10 μl of 10 × PCR buffer (Cina gene, Iran), 2003). The nuclear ITS is used more commonly 1.5 mM MgCl2, 20 pg of each primer and 2.5 U of Taq among Gyrodactylus species (Kuusela et al., 2008). polymerase (Fermentas) in a total volume of 100 µl. It is thought that the combination of morphological The amplification was carried out in a thermocycler analysis with a molecular method can result in the (MWG, Germany) using step one at 95°C for 5 beter method to identify the Gyrodactylus species. minutes; step two with 38 cycles, at 94°C for 45 This paper presents the identification of Gyrodactylus seconds; 50°C annealing temperature for 45 seconds, species on Carassius auratus using both morphological 72°C for 45 seconds; and, the final step taken for 10 and molecular characteristics. minutes at 72 °C. The PCR products were analyzed on 1.5% agarose gel and visualized under the UV Materials and Methods illuminator and then the results were recorded. PCR product purification and sequencing:The Sampling and microscopic examination: In the PCR products were purified using a PCR purification present study, a total of fifty goldfish (Carassius kit (MBST, Iran) following the manufacturer's auratus) were referred alive in their original water, to instructions. Purified fragments were sequenced the Aquatic Animal Health Department laboratory from both sites of each PCR product using a method (Faculty of Veterinary Medicine, University of based on Sanger (1977). Sequencing was carried out Tehran, Tehran, Iran). The body and fins of each fish using the same primers as used for PCR amplification, were immediately examined by wet mounts, by kowsar company. microscopic slides were prepared from parasite samples and the existence of Gyrodactylus was Results analyzed using a light microscope. Each parasite was separately photographed by digital camera (Sony, Gyrodactylus parasites were identified in 15 out 42 IJVM (2012), 6(1):41-46 Ebrahimzadeh Mousavi, H.A. Identification of Gyrodactylus gurleyi in... Table 1. Morphological measurements of opisthaptoral hard parts of Gyrodactylus spp. collected from Carassius auratusin present study and Gyrodactylus gurleyi reported by Ergens &Yukhimenko (1987) and Cones &d Wiles (1983). Measurement (μm) Present study Ergens andYukhimenko(1987) Cones and Wiles(1983) Total length of anchor 47.55-52.96 45-55 47-56 Length of anchor root 10.07-17.34 13-19 10-18 Length of anchor shaft 35.72-40.75 33-42 33-43 Length of anchor point 21.65-26.15 23-28 21-27 Marginal hook sickle length 4.98-5.11 5 5 Marginal hook handle length 21.77-22.3 - 22 Total length of marginal hook 24.22-27.5 23-28 24-28 Length of ventral bar 19.81-21.74 18-22 18-25 Median width of ventral bar 3.75-5.03 5-6 3-5 length of ventral bar membrane 10.26-12.52 10-13 9-14 Total length of dorsal bar 19.42-23.14 12-18 19-22 Median width of dorsal bar 1.99-2.15 1-2 - Figure 1: Opisthaoptoral hard parts characters of Gyrodactylus gurleyi used for morphological analysis in present study: (A) Anchor: (1) total length of anchor; (2) length of anchor shaft; (3) length of anchor root; (4) length of anchor point. (B) Marginal hook: (5) length of sickle; (6) length of handle; (7) total length of marginal hook; (C) Ventral bar: (8) median width of ventral bar; (9) length of ventral bar; (10) length of ventral bar membrane; (D) Dorsal bar: (11) median width of dorsal bar; (12) total length of dorsal bar. of the 50 fish samples. Gyrodactylus specimens 47.55-52.96; Length of anchor root 10.07-17.34; collected from goldfish samples were identified as Length of anchor shaft 35.72-40.75; Length of Gyrodactylus gurleyi by both morphometric and anchor point 21.65-26.15; Total length of marginal molecular analysis. hook 24.22-27.5; Marginal hook sickle length 4.98- Morphological description:The morphological 5.11; Marginal hook handle length 21.77-22.3, characteristics of Gyrodactylus samples were Length of ventral bar 19.81-21.74; Median width of compared with available Gyrodactylus morphology.
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